WO2000073439A1 - Pollinosis-associated gene 465 - Google Patents

Abstract

A novel gene undergoing significantly lowered expression after the pollen scattering has been successfully isolated by preparing T cells from subjects before and after the pollen-scattering season and searching the gene by the differential display method. It is found out that this gene is usable in examining allergic diseases and screening candidate compounds for remedies capable of regulating the response of T cells to the stimulus by an antigen.

Description

 Pollen alleles, 465 technical fields

 The present invention relates to a gene associated with an antigen stimulatory response, a method for testing an allergic disease using the expression of the gene as an index, and a method for screening a candidate therapeutic compound for suppressing a T cell antigen stimulatory response. Background art

 Allergic diseases, including hay fever, are considered multifactorial diseases. These diseases are caused by the interaction of the expression of many different genes, and the expression of these individual genes is affected by multiple environmental factors. Therefore, it is very difficult to elucidate the specific genes that cause specific diseases.

 It is thought that the onset of allergic disease involves mutations or defects in genes expressed in response to antigenic stimulation, or overexpression or decreased expression levels. To understand the role of gene expression in disease, it is necessary to understand how genes are involved in pathogenesis and how external stimuli, such as antigens and drugs, alter gene expression.

Recent advances in gene expression analysis technology have made it possible to analyze and compare gene expression in many clinical samples. The differential display (DD) method is useful as such a method. The differential display method was first developed in 1992 by Liang and Pardee (Science, 1992, 257: 967-971). By using this method, dozens or more of samples can be screened at a time, and It is possible to detect a gene whose expression has changed. Using such a method to examine genes with mutations or genes whose expression changes with time or environment is expected to provide important information for elucidating pathogenic genes. These genes include those whose expression is affected by environmental factors.

 Allergic diseases such as hay fever are one of the diseases seen by many people in recent years. The pathogenesis of hay fever may be related to several genes whose expression is affected by pollen, one of the environmental factors. Under such circumstances, it has been desired to isolate genes associated with allergic diseases such as hay fever. Disclosure of the invention

 An object of the present invention is to provide a gene associated with an allergic disease. Further, the present invention provides a method for testing an allergic disease, using the expression of the gene as an index.

It is an object of the present invention to provide a method for screening a candidate therapeutic compound that suppresses the T cell antigen-stimulated response.

 The present inventors have proposed a method for treating a plurality of humans based on the already established procedure of the “fluorescent DD (Fluorescent DD) method” (T. I. to et al., 1994, FEBS Lett. 351: 231-236). We developed a new DD system that can analyze T-cell RNA samples prepared from human blood. Using this system, the present inventors collected T cells from blood before and after pollen dispersal for a plurality of subjects including pollinosis patients. We screened for genes whose expression levels change, and isolated a novel gene (the “465” gene).

The present inventors have compared and analyzed the expression level of the isolated `` 465 '' gene in lymphocytes isolated from subjects before and after pollen scattering, and found that the gene showed a significantly lower value after cedar pollen scattering. Was found. Therefore, the present inventors have examined the allergic disease and the treatment for suppressing the antigen stimulation response of T cells using the expression level of the gene as an indicator. It has been found that screening of drug candidate compounds is possible.

 That is, the present invention relates to a gene exhibiting a low value after pollen scattering, a method for testing an allergic disease using the expression of the gene as an index, and a method for screening a candidate compound for a therapeutic agent which suppresses antigen stimulation response of T cells. More specifically,

 [1] a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 23,

 [2] a nucleic acid molecule comprising the protein coding region of the nucleotide sequence of SEQ ID NO: 23, [3] a strand of at least 15 nucleotides which specifically hybridizes to the nucleic acid molecule of [1] or [2]; DNA having a length,

 (4) The method for detecting a nucleic acid molecule according to (1), which comprises using the DNA according to (3).

 [5] a method for testing an allergic disease,

 (a) preparing T cells from a subject,

 (b) preparing an RNA sample from the T cells,

 (c) a step of performing hybridization on the RNA sample using the labeled DNA according to (3) as a probe,

 (d) measuring the amount of RNA derived from a subject that hybridizes to the labeled DNA according to [3], and comparing it with a control (in the case of a healthy subject).

 [6] a method for testing an allergic disease,

 (a) preparing T cells from a subject,

 (b) preparing an RNA sample from the T cells,

 (c) performing a reverse transcription reaction on the RNA sample to synthesize cDNA;

 (d) a step of performing a polymerase chain reaction (PCR) using the cDNA as a type III and the DNA according to [3] as a primer,

 (e) comparing the amount of MA amplified by the polymerase chain reaction with a control (in the case of a healthy subject).

[7] The method according to [6], wherein the polymerase chain reaction is performed by a PCR amplification monitor method. Law,

 (8) the method according to any of (5) to (7), wherein T cells are prepared from peripheral blood of the subject;

 (9) the method according to any one of (5) to (8), wherein the allergic disease is cedar pollinosis;

 [10] 方法 a method of screening for a candidate therapeutic compound that suppresses the antigen-stimulated response of cells,

 (a) Step of administering a test compound to a model animal and stimulating it with a pollen antigen

(b) preparing T cells from the model animal,

 (c) preparing an RNA sample from the T cells,

 (d) performing a hybridization on the RNA sample using the labeled DNA according to (3) as a probe,

 (e) measuring the amount of RNA derived from the T cells that hybridizes to the labeled DNA according to (3),

 (f) selecting a compound that suppresses the decrease in the amount of RNA measured in step (e) as compared to a control (in the case where the test compound is not administered);

 (11) A method for screening a candidate therapeutic compound that suppresses the antigen-stimulated response of T cells,

 (a) administering a test compound to a model animal and stimulating with a pollen antigen,

(b) preparing T cells from the model animal,

 (c) preparing an RNA sample from the T cells,

 (d) performing a reverse transcription reaction on the RNA sample to synthesize cDNA;

 (e) a step of performing a polymerase chain reaction (PCR) using the cDNA as a type III and the DNA according to [3] as a primer,

(f) selecting a compound that suppresses a decrease in the amount of DNA amplified in step (e) compared to a control (in the case where the test compound is not administered), (12) A method for screening a candidate therapeutic compound that suppresses the antigen stimulation response of T cells,

 (a) administering a test compound to a model animal,

 (b) preparing lymphocytes from the model animal,

 (c) stimulating the lymphocytes with a pollen antigen,

 (d) separating T cells from the antigen-stimulated lymphocytes,

 (e) preparing an RNA sample from the T cells,

 (f) a step of performing hybridization on the RNA sample using the labeled DNA of (3) as a probe,

 (g) a step of measuring the amount of RNA derived from the T cells that hybridizes to the labeled DNA according to (3),

 (h) selecting a compound that suppresses the decrease in the amount of RNA measured in step (g) compared to a control (in the case where the test compound is not administered),

 (13) a method for screening a candidate compound for a therapeutic agent that suppresses the antigen stimulation response of T cells,

 (a) administering a test compound to a model animal,

 (b) preparing lymphocytes from the model animal,

 (c) stimulating the lymphocytes with a pollen antigen,

 (d) separating T cells from the antigen-stimulated lymphocytes,

 (e) preparing an RNA sample from the T cells,

 (f) performing a reverse transcription reaction on the RNA sample to synthesize cDNA;

 (g) a step of performing a polymerase chain reaction (PCR) using the cDNA as a type III and the DNA according to [3] as a primer,

 (h) selecting a compound that suppresses a decrease in the amount of DNA amplified in step (g) as compared to a control (in the case where the test compound is not administered);

[14] Screen for candidate therapeutic agents that suppress T cell antigen stimulation response Method

 (a) preparing lymphocytes from a model animal or human mosquito,

 (b) stimulating the lymphocytes with a pollen antigen in the presence of a test compound,

 (c) separating T cells from the antigen-stimulated lymphocytes,

 (d) preparing an RNA sample from the T cells,

 (e) performing a hybridization on the RNA sample using the labeled DNA according to (3) as a probe,

 (f) measuring the amount of RNA derived from the T cells that hybridizes to the labeled DNA according to (3),

 (g) selecting a compound that suppresses the decrease in the amount of RNA measured in step (f) as compared to a control (in the case where the test compound is not administered);

 (15) a method of screening for a therapeutic candidate compound that suppresses the antigen-stimulated response of T cells,

 (a) preparing lymphocytes from a model animal or human,

 (b) stimulating the lymphocytes with a pollen antigen in the presence of a test compound,

 (c) separating T cells from the antigen-stimulated lymphocytes,

 (d) preparing an RNA sample from the T cells,

 (e) performing a reverse transcription reaction on the RNA sample to synthesize cDNA;

 (f) performing a polymerase chain reaction (PCR) using the cDNA as a type I and the DNA according to [3] as a primer,

 (g) selecting a compound that suppresses a decrease in the amount of DNA amplified in step (f) as compared to a control (in the case where the test compound is not administered);

 (16) a method for screening a candidate therapeutic compound that suppresses the antigen stimulation response of T cells,

 (a) stimulating the established T cells with a lymphocyte stimulating substance in the presence of the test compound,

(b) preparing an RNA sample from the stimulated established T cells, (c) a step of subjecting the DNA according to (3) to a hybridization with the RN ^; A sample,

 (d) measuring the amount of RNA derived from the established T cells that hybridizes to the labeled DNA according to (3),

 (e) selecting a compound that suppresses the decrease in the amount of RNA measured in step (d) compared to a control (in the case where the test compound is not administered),

 (17) a method for screening a candidate therapeutic compound that suppresses the antigen stimulation response of T cells,

 (a) stimulating the established T cells with a lymphocyte stimulating substance in the presence of the test compound,

(b) preparing an RNA sample from the stimulated established T cells,

 (c) performing a reverse transcription reaction on the RNA sample to synthesize cDNA;

 (d) a step of performing a polymerase chain reaction (PCR) using the cDNA as a type I and the DNA according to [3] as a primer,

 (e) selecting a compound that suppresses a decrease in the amount of DNA amplified in step (d) compared to a control (in the case where no test compound is administered),

 (18) T cells are prepared from peripheral blood of a model animal, (10) or the method according to (11),

 (19) the method of any one of (12) to (15), wherein the lymphocytes are prepared from peripheral blood,

 (20) the method according to any one of (10) to (19), wherein the antigen is a cedar pollen antigen,

 About.

In the present invention, allergic disease is a general term for diseases associated with allergic reactions. More specifically, it can be defined as identifying the allergen, demonstrating a deep link between exposure to the allergen and the development of the lesion, and demonstrating an immunological mechanism for the lesion. Here, the immunological mechanism is the allele Means that T cells show an immune response upon the stimulation of the gene. Representative allergic diseases can include bronchial asthma, allergic rhinitis, atopic dermatitis, hay fever, or insect allergy. Allergic predisposition (all ergi cdi athes is) is a genetic factor transmitted from parents to children with allergic diseases. Allergic diseases that occur familially are also called atopic diseases, and the genetic factors that cause them are atopic predisposition.

 The “nucleic acid molecule” in the present invention includes DNA and RNA.

 The present invention relates to a novel gene “465” that correlates with the response of lymphocytes to cedar pollen antigen. The nucleotide sequence of "465" cDNA found by the present inventors is shown in SEQ ID NO: 23.

 The nucleotide sequence of the “465” cDNA isolated by the present inventors is a partial distribution sequence of the “465” cDNA, and those skilled in the art will understand that the sequence information of the “465” cDNA described in SEQ ID NO: 23 is Isolation of full-length cDNA of “465” based on the information can be usually performed. That is, a method for screening a T cell cDNA library or the like by hybridization using a sequence derived from “465” as a probe, or a method for screening a T cell cDNA library using a sequence derived from “465” as a primer. There is a method of obtaining the full-length cDNA by screening a library using such DNA as type II and obtaining an amplification product having a size specific to the primer as an index. The RACE method (Frohman, MA et al .: Using the sequence derived from “465” as a primer, converting mRNA from T cells and the like into single-stranded cDNA, adding an oligomer to the end, and then performing PCR. Proc. Natl. Acad. Sci. USA, 85: 8992, 1988).

 The “nucleic acid molecule comprising the base sequence of SEQ ID NO: 23” in the present invention includes “46” which can be isolated based on the sequence information of “465” cDNA of SEQ ID NO: 23 as described above. 5 "full-length cDNA.

“465” showed statistically significantly lower expression of lymphocytes in subjects after exposure to pollen antigen than before exposure. Therefore, expression of the “465” gene (conversion to mRNA) (Including transcription and translation into proteins) as indicators, it will be possible to test for allergic diseases and to screen for candidate therapeutic compounds that suppress antigen-stimulated response of T cells. Decreased expression of “465” indicates the response of T cells to the stimulation of antigens such as pollen.In patients with a history of allergic disease, the expression of “465” is used as an index to determine the response of the patient to specific antigen exposure. Monitoring the transition is helpful in understanding the state of the disease and examining response to treatment.

 As the allergic disease to be tested and treated in the present invention, cedar pollinosis is particularly preferred.

 The detection of the expression of the `` 465 '' gene in the test for allergic disease according to the present invention can be performed by a hybridization technique using a nucleic acid that hybridizes to the `` 465 '' gene as a probe, or a DNA hybridizing to the gene of the present invention as a primer. It is possible to carry out using the gene amplification technology described above.

 As the probe or primer used in the test of the present invention, a nucleic acid molecule that specifically hybridizes to the “465” gene and has a chain length of at least 15 nucleotides is used. The term "specifically hybridizes" as used herein refers to a DNA that is cross-hybridized with DNA and Z or RNA encoding another gene under ordinary hybridization conditions, preferably under stringent hybridization conditions. Refers to no significant occurrence. For example, the probe and transfer membrane were hybridized at 68 in Expres s Hydidi zat ion on So luti on (manufactured by CL0NTECH), and finally, 0.1 X SSC, 0.05% SDS solution By washing with 50 at, stringent conditions can be achieved.

These nucleic acid molecules used in the test of the present invention may be synthetic or natural. The probe DNA used for hybridization is usually labeled. Labels include, for example, labeling with nick transduction using DNA polymerase I, end labeling using polynucleotide kinase, fill-in labeling using Klenow fragment (Berger SL, Kimmel AR. (1987) Guide to Molecular Cloning Techniques, Method in Enzymology, Academic Press; Hames BD, Higgins SJ (1985) Genes Probes: A Practical Approach.IR L Press; Sambrook J, Fritsch EF, Maniatis T. (1989) Molecular Cloning: a Laboratory Manual, 2nd Edn. Cold Spring Harbor Laboratory Press), Labeling by transcription using RNA polymerase (Melton DA, Krieg, PA, Rebagkiati MR, Maniatis T, Zinn K, Green MR. (1984) Nucleic Acid Res., 12 , 7035-7056), and a method of incorporating a modified nucleotide without using a radioisotope into DNA (Kricka LJ. (1992) Nonisotopic DNA Probing Techniques. Academic Press). Testing of allergic diseases using the hybridization technology can be performed using, for example, a Northern hybridization method, a dot blot method, a method using a DNA microarray, or the like.

 On the other hand, as a method using gene amplification technology, for example, an RT-PCR method can be used. In the RT-PCR method, if the PCR amplification monitor method is used as shown in Example 8 in the gene amplification process, more accurate quantification of the expression of the “465” gene can be performed.

In the PCR gene amplification monitoring method, probes that are labeled with different fluorescent dyes at both ends to cancel each other's fluorescence are used to hybridize to the detection target (reverse transcript of DNA or RNA). When the PCR proceeds and the probe is degraded by the 5'-3 'exonuclease activity of Taq polymerase, the two fluorescent dyes separate and the fluorescence is detected. This fluorescence is detected in real time. The number of copies of the target in the target sample is determined based on the number of linear cycles of PCR amplification by simultaneously measuring a standard sample with a clear copy number for the target (Holland, PM et al., 1991, Pro Natl. Acad. Sci. USA 88:72 76-7280; Livak, KJ et al., 1995, PCR Methods and Applications 4 (6): 35 7-362; Heid, CA et al., Genome Research 6: 986. -994; Gibson, EMU et al., 1996, Genome Research 6: 995-1001). In the PCR amplification monitoring method, For example, ABI PRI SM7700 (PerkinElmer) can be used.

 In addition, the test for an allergic disease of the present invention may be performed by detecting the protein encoded by “465”. As such a test method, for example, a Western blotting method using an antibody that binds to the protein encoded by “465”, an immunoprecipitation method, an ELISA method, and the like can be used. The antibody of the protein encoded by "465" of the present invention can be obtained as a polyclonal antibody or a monoclonal antibody using techniques well known to those skilled in the art (Milstein in C, eta, 1983, Nature 305). (5934): 537-40). For example, a protein or a partial peptide thereof used as an antigen is prepared by incorporating the "465" gene or a part thereof into an expression vector, introducing this into an appropriate host cell, and preparing a transformant. Is cultured to express a recombinant protein, and the expressed recombinant protein is purified from a culture or a culture supernatant. As a result of the test for an allergic disease according to the present invention, if the expression of the gene of the present invention is significantly low, it can be determined that the subject has developed an immune response to an allergen such as cedar pollen antigen. The measurement of the expression level of the gene of the present invention, together with the pollen-specific antibody titer, symptoms, etc., can be used for testing for allergic diseases.

The expression of the “465” gene expressed in T cells is reduced after pollen antigen exposure. The “465” gene is a gene whose expression level decreases as a response of the living body to the cedar pollen antigen stimulation, and screening of the remedy for hay fever can be performed by monitoring the expression of the “465” gene. The expression level of the “465” gene is reduced by pollen antigen exposure in both healthy and hay fever patients. Presence or absence of hay fever symptoms is presumed to be due to differences after the response of "465J gene" to antigen stimulation. However, even in such cases, decreased expression of "465" gene corresponds to enhanced T cell responsiveness Therefore, screening of therapeutic agents for allergic diseases can be performed by monitoring the expression of the “465” gene. The method of the present invention for screening a therapeutic drug candidate compound that suppresses antigen-stimulated response of T cells can be performed in vivo or in vitro. In in vivo screening, for example, after administering a candidate drug and stimulating with a pollen antigen to a model animal such as a mouse, T cells are separated from peripheral blood, and the transcript of “465” is measured. . Alternatively, after administering the candidate drug to a model animal such as a mouse, lymphocytes are separated from peripheral blood, and the lymphocytes are stimulated in vitro with cedar pollen antigen or the like. T cells are separated from the lymphocytes after the stimulation, and the transcript of the “465” gene is measured. As a result of these measurements, a compound that suppresses a decrease in the transcription level of the “465” gene compared to a control (when no candidate drug is administered) is selected. Here, the stimulation with the pollen antigen is performed for the purpose of eliciting an antigen-specific allergic reaction in T cells and determining the therapeutic effect of the candidate compound on it. In the present invention, suppressing the decrease in the transcription amount of the “465” gene means that a higher level of transcription amount is maintained by contact with a candidate compound when compared with antigen-stimulated T cells. Say Therefore, if the candidate compound induces a level that exceeds the transcript level of the “465” gene before being subjected to antigenic stimulation (ie, if transcription is increased), the compound is a compound to be selected in the screening method of the present invention. .

 In the in vitro screening, for example, peripheral blood lymphocytes are collected from a human mouse or the like, and the peripheral blood lymphocytes are stimulated in vitro with cedar pollen antigen or the like. Candidate compounds are added during in vitro stimulation. Then, T cells are separated from the stimulated peripheral blood lymphocytes, and the “465” transcript is measured. As a result of this measurement, a compound that suppresses a decrease in the transcription amount of the “465” gene as compared to a control (when no candidate drug is contacted) is selected.

Screening of the candidate therapeutic compound for suppressing the antigen-stimulated response of T cells of the present invention can also be performed using established T cells. For example, established T cells such as Mol cells and kaurka t cells are stimulated in vitro with a lymphocyte stimulator. Lymphocyte stimulants include, for example, calcium ionophore (A23187), PMA, Glutinin (PHA) and the like. Add candidate drug during in vitro stimulation. Thereafter, the transcription amount of the “465” gene in the established T cells is measured. As a result of this measurement, a compound that suppresses a decrease in the transcription amount of the “465” gene as compared to a control (when no candidate drug is contacted) is selected.

 Detection of the expression of the "465" gene in the screening of a therapeutic drug candidate compound that suppresses the T cell antigen-stimulatory response according to the present invention can be performed by detecting the expression of the "465" gene in the same manner as the test for an allergic disease of the present invention. The hybridization can be carried out using a hybridization technique using DNA as a probe, or a gene amplification technique using a DNA that hybridizes to the gene of the present invention as a primer.

 As a method using the hybridization technology, for example, a Northern hybridization method, a dot blot method, a method using a DNA microarray, or the like can be used. On the other hand, as a method utilizing the gene amplification technique, an RT-PCR method can be used. In the RT-PCR method, more accurate quantification of the expression of the “465” gene can be performed by using a PCR amplification monitoring method as shown in Example 8 in the gene amplification process.

 The test compounds used in these screenings include compound samples synthesized by existing chemical methods such as steroid derivatives, compound samples synthesized by combinatorial chemistry, and extracts of animal and plant tissues. Alternatively, a mixture containing a plurality of compounds such as a microorganism culture, and a sample purified therefrom may be mentioned. The compound isolated by the method of the present invention for screening a candidate therapeutic compound for suppressing a T cell antigen-stimulated response is a candidate for a drug that suppresses an immune response.

When the compound isolated by the screening method of the present invention is used as a pharmaceutical, it can be used as a pharmaceutical preparation by a known pharmaceutical production method. For example, it is administered to a patient together with a pharmacologically acceptable carrier or vehicle (such as saline, vegetable oils, suspensions, surfactants, stabilizers, etc.). Administration will be transdermal, intranasal, transbronchial, intramuscular, intravenous, or oral, depending on the nature of the compound. The dose varies depending on the patient's age, body weight, symptoms, administration method and the like, but those skilled in the art can appropriately select an appropriate dose. BRIEF DESCRIPTION OF THE FIGURES

 FIG. 1 is a diagram showing the antibody titers of cedar pollen-specific IgE antibodies in a total of 18 blood samples from 10 subjects who collected blood. The values of cedar pollen-specific IgE antibodies in each blood sample of Test Subjects A to (sample numbers 1 to 18) were expressed in AU / ml. The pair before pollen scattering is shown on the left (white column), and the one after scattering is shown on the right (black column). Subjects A and B collected only blood after pollen scattering.

 FIG. 2 is a diagram showing changes in the expression of “465” in the pre-scattering group and the post-scattering group when the groups are divided according to the time before and after the cedar pollen scattering. Error bars represent standard deviation. FIG. 3 is a photograph showing the results of Northern hybridization of “465” using mRNA prepared from various immune-related tissues.

 FIG. 4 is a photograph showing the results of Northern hybridization of “465” using mRNA prepared from various cancer cell lines. BEST MODE FOR CARRYING OUT THE INVENTION

 Hereinafter, the present invention will be described specifically with reference to Examples, but the present invention is not limited to these Examples.

 [Example 1] Blood collection from 10 adult volunteers

Blood samples of 10 adult volunteers (A ~; 0 to 10 ml) were collected before and after pollen dispersal to collect T cells before and after pollen dispersal. The first blood sample was Japanese cedar pollen dispersal Before the season of January, January and February of 0997), and the second after the Japanese cedar pollen was dispersed (March, April and May of 1997). For eight of the volunteers, samples from two periods were obtained. For the remaining two volunteers, only samples after pollen scattering were available. One of these blood samples The amount of the cedar pollen-specific IgE was measured using the test pieces. The specific IgE is measured by the CAP RAST method (Pharmacia), which is an improved version of the RAST method (radio allergo sorbent test, Wide, L. et. Al .: Lancet 2: 1105-1107, 1967) using a paper disk as a solid phase. I went. Using a serum containing a standard antibody titer manufactured by Pharmacia, and using that as a reference, the IgE antibody titer of each sample (the unit is also indicated as Pharmacia RAST Unit, PRU, or AU (arbitrary unit)) It was determined.

 Fig. 1 shows the measured cedar pollen-specific IgE values before and after pollen scattering in each subject. As shown, most of the 10 subjects had increased serum levels of cedar pollen-specific IgE after pollen exposure. The presence of atopic predisposition was determined by whether the value of the CAP RAST test for cedar pollen-specific IgE was greater than 2. That is, eight subjects A to G and I were considered to be atopic predisposition groups (hereinafter also referred to as “patients”), and two subjects H and were considered to be healthy subjects (hereinafter also referred to as “normal groups”). Of the eight subjects with an atopic predisposition, seven exhibited symptoms of allergic rhinitis after pollen dispersal.

 [Example 2] Preparation of lymphocyte fraction from blood sample

When preparing T cells from 10 ml of blood, the procedure was as follows. First, the wall of the syringe was treated with 1 ml of Heparin from Nopo, etc. without any restriction, and blood was collected in a 10 ml syringe containing a final concentration of 50 unit / ml heparin. At this time, two 22G needles were prepared for one blood sample. The injection needle was removed and transferred to a 50 ml centrifuge tube (made of polypropylene). After centrifugation at 1500 rpm for 5 minutes at room temperature, 1.1 ml was collected from as close to the surface as possible, and centrifuged at 15000 rpm for 5 minutes at 4 to collect 1 ml of the supernatant as plasma. An equal volume (9 ml) of 0.9% NaCl containing 3% dextran (manufactured by Nacalai) was added to the rest of the collected plasma, and the mixture was gently inverted several times to mix. Then, it was left still at room temperature for 30 minutes. PRP (Pla teletrich plasma, platelet-rich plasma) was transferred to another 15 ml centrifuge tube, and centrifuged at 1200 rpm (equivalent to 150 Xg in a centrifuge manufactured by Tomi) at room temperature for 5 minutes. After centrifugation, platelets were in the supernatant. Ca, Mg obtained from the precipitated cells from Gibco It was suspended in 5 ml of free HBSS. This was overlaid on one tube (Falcon tube: 2006 or 2059; made of polypropylene) containing 5 ml of Ficol Paque (Pharmacia) using a Pasteur pipette. After centrifugation at 1200 ι · ριη for 5 minutes, the mixture was centrifuged at 1500 rpm (equivalent to 400 Xg in a Tomy centrifuge) at room temperature for 30 minutes. As a result, granulocytes and erythrocytes precipitated, and lymphocytes, monocytes, and platelets were contained in the middle layer with the ficoll layer between them. Collect the intermediate layer with a Pasteur pipette, add 2 to 3 volumes of BSA / PBS (0.5% BSA, 2π EDTA in PBS, pH 7.2; degas immediately before use), and add 1200 rpm, 4 Centrifuged at ° C for 5 minutes. The precipitate was collected and washed twice with BSA / PBS. After the second washing, the cells were suspended in 5 ml, and a part thereof was diluted 2-fold with trypan blue, and the number of cells was counted. Total cell number was about IX 10 ^7. This was used as the lymphocyte fraction.

 [Example 3] Separation of T cells from lymphocyte fraction

The lymphocyte fraction obtained in Example 2 was centrifuged at 1200 rpm at 4 ° C. for 5 minutes, and suspended in BSA / PBS at a concentration of 10 ⁸ per 100 / il. The capacity became about 201. This was transferred to an Eppendorf tube (1.5 ml), and the CD3 microbead solution was added. Then, it was left at 4-10 for 30 minutes (at this time, it was not placed on ice). This sample was treated with Magnetic Celso Ichiichi (MACS) (Miltenyi Biotech In) as follows.

 The MS + / RS + column was attached to a Mini MACS or Vario MACS separation unit (without needles). 500 Ml of BSA / PBS was gently applied to the column and the buffer was drained. Next, cells labeled with CD3 microbeads were applied to the column. The column was washed three times with 500 / zl (B cell fraction). The column was removed from the separation unit and placed on a tube for collecting the eluate. 1 ml of BSA / PBS was applied to the column, and positive cells were rapidly flushed out using a plunger attached to the column. This was used as the T cell fraction.

The obtained T cell fraction was centrifuged at 1200 rpm for 5 minutes at 4 ° C. The precipitate was washed twice with BSA / PBS. After the second wash, resuspend the cells in 1 ml and partially remove The number of cells was measured by diluting with blue two-fold. Total cell number was approximately 4 × 10 ⁶ .

 [Example 4] Preparation of total RNA from T cells

Preparation of total RNA from T cells was performed using RNeasy Mini (Qiagen) according to the attached manual in principle. All operations were performed at room temperature, wearing gloves. Four times the volume of ethanol was added to Posh Buffer-RPE. The lysis buffer RLT was supplemented with Ιθΐ / ml of 2-mercaptoethanol. The cell suspension was centrifuged at 1000-1200 rpm for 5 minutes, and the supernatant was removed by evaporation. To the precipitate was added 350 il lysis buffer RLT (containing 2-mercaptoethanol) solution. At this stage, lysates of cells in RLT buffer could be stored at -70 ° C. If the cell lysate had been stored frozen, incubate at 37 for 10-15 minutes, and if insoluble material was visible, centrifuge at maximum speed for 3 minutes, collecting only the supernatant. The lysate was homogenized with a syringe equipped with a 20 G force teran needle, and then treated with QIAshredder. (That is, usually a 350/1 cell lysate was applied to a Kyaschlets unit using a Pitman. The mixture was centrifuged at 1500 μηι for 2 minutes, and the effluent was collected.) 70% of 350 1 Ethanol was added and mixed well by pipetting. The RNeasy spin column was attached to the attached 2 ml tube, the cell lysate mixture was applied, centrifuged at 8000 X g (1 1500 rpm) for 1 minute, and the effluent was discarded. Posh buffer RW1 700 1 was applied to the column, and it was set up with a lid for 5 minutes. 1 Centrifuged at 1500 rpm for 15 seconds and discarded the effluent. The column was placed in a new 2 ml tube, and Posh Buffer RPE (containing ethanol) 500 1 was applied to the column, followed by centrifugation at 1500 rpm for 15 seconds, and the effluent was discarded. The wash buffer RPE5001 was applied to the column and centrifuged at maximum speed for 2 minutes. The column was placed in a new 1.5 ml tube, 30/1 DEPC-treated water was applied, and the lid was capped and allowed to stand for 10 minutes. 1 Centrifuged at 1500Ι for 10 minutes to obtain total RNA. Measure the concentration, and if the volume is low, re-attach the column to a new 1.5 ml tube, apply DEPC-treated water, close the lid, stand for 10 minutes, and set the column at 1500 i "pm. Centrifuge for minutes.

 [Example 5] DNase treatment of total RNA

 DNase treatment was performed to remove DNA from total RNA prepared from T cells. Reactions were performed in 100 1 1 XDNase buffer (Futsuba Gene) containing 2 units of DNase (Futsuba Gene) and 50 units of RNase Inhibitor Yuichi (Pharmacia). After incubating this at 37 ° C for 15 minutes, an equal amount of PCI (phenol: black form: isoamyl alcohol = 25: 24: 1) was added, and the mixture was portexed. The mixture was centrifuged at 12,000 rpm at room temperature for 10 minutes, and the upper layer (aqueous layer) was transferred to a new 1.5 ml tube. One-tenth volume of 3M sodium acetate (pH 5.2) was added, and 2.5 volumes of 100% ethanol and eta-precipitated mate 1 ^ 1 were added and mixed by inversion. After allowing to stand at -20 ° C for 15 minutes, the mixture was centrifuged at 12000 rpm for 4 and 15 minutes, the supernatant was removed, and 70% ethanol was added. After tapping to the extent that the precipitate was detached, the supernatant was removed cleanly. Dried for 3 minutes and dissolved in 10-201 DDW (no DNase and RNase). The concentration was measured and stored at -80 ° C until use.

 [Example 6] Differential display (DD) analysis using total RNA prepared from T cells

 The analysis of fluorescent differential display (F1 uorescent Differential Display, abbreviated as “DD”) using total RNA prepared from T cells is described in the literature (T. Ito et al., 1994, FEBS Lett. 351: 231-236). Performed according to the method. Total RNA prepared from T cells was reverse transcribed to obtain cDNA. For the primary DD-PCR reaction, cDNA was prepared using 0.2 // g of total RNA for each of the three anchor primers. For the secondary DD-PCR reaction, cDNA was prepared using RNA 0.4 / zg for each of the three anchor primers. Each cDNA was diluted to a final concentration equivalent to 0.4 ng / 1 RNA and used for the experiment. A DD-PCR reaction was performed using cDNA equivalent to 1 ng RNA per reaction. Table 1 shows the composition of the reaction solution.

table 1 cDNA (0.4ng // x 1 RNA equivalent) 2.5 / 1

 Optional primer 2.5 ^ 1

 lOXAmpliTaq PCR buffer-1.0 1

 2.5mM dNTP 0.8 1

50 M anchor primer 0.1 μ 1

 (GT15A, GT15C, GT15G)

 Gene Taq (5U / 1) 0.05w

 Am l iTaq (5U // 21) 0.05

dH ₂ 0 3.0 1

10.0M

The PCR reaction conditions were as follows: `` 95 ° C for 3 minutes, 40 ° C for 5 minutes, 72 ° C for 5 minutes '' for one cycle, followed by `` 94 for 15 seconds, 4 (TC2 minutes, 72 ° C for 1 minute) for 30 cycles. Thereafter, the temperature was changed to 72 ° C for 5 minutes, and then continuously to 4. The primer pairs used were the anchor-primers GT15A (SEQ ID NO: 2), GT15C (SEQ ID NO: 3), and GT15G (SEQ ID NO: 4). ) Were combined with arbitrary primers AG 1 to 110, AG 111 to 199, and AG 200 to 287, respectively, and a total of 287 sets of reactions were performed. Oligomers composed of nucleotides were designed, synthesized and used.

 For gel electrophoresis, a 6% denaturing polyacrylamide gel was prepared, a 2.5 ^ 1 sample was applied, and electrophoresed at 40 W for 210 minutes. Thereafter, the gel plate was scanned using Hitachi Fluorescence Image Analyzer -FMBI0 II, and electrophoretic images were obtained by fluorescence detection.

 [Example 7] Amplification and sequencing of band cut out by DD analysis

Two DD analyzes were performed using a number of arbitrary primers. Select a band that has a difference between before and after pollen scattering or between the patient and healthy group, One band was cut out of the gel.

 Further analysis was performed on one of the cut bands (referred to as "465"). The band of “465” was found by DD analysis using GT15A (SEQ ID NO: 2) as an anchor primer and AG75 (AGCTMGAGG / SEQ ID NO: 5) as an arbitrary primer.

 To determine the nucleotide sequence of “465”, a gel containing the “465” band was cut out, stored in a TE solution, and heated at 60 ° C. for 10 minutes to elute DNA from the gel. Using this TE solution as type II, KR was performed under the same conditions as DD-PCR, and a DNA fragment of about 270 bp was amplified. GT15A was used as the primer and AG75 was used as the optional primer. The amplified DNA fragment was cloned with a plasmid vector pCR2.l (Invitrogen) to obtain a plasmid p465-16 holding a DNA fragment of about 270 bp. Using the plasmid DNA, the nucleotide sequence of the DNA fragment was determined according to a conventional method.

 [Example 8] Quantification by ABI-7700

 The expression amount of “465” was quantified by the TaqMan method using ABI-PRI SM7700. This method uses a fluorescent dye to quantitatively detect the PCR-amplified DNA strand in real time. For the purpose of quantification, in the spring of 1998, a new blood sample before and after the cedar pollen was dispersed was collected from 22 volunteers, T cells were prepared, and total RNA was extracted. The expression level of the target gene was quantified using a total of 44 total RNA samples.

In the same manner as in Example 1, specific IgE values and total IgE values of cedar pollen, cypress pollen, Dermatophagoides farinae, and Dermatophagoides farinae were measured (Table 2). Table 2

Specific IqE 值

 Specific lgE (UA / mi)

 Subject Blood collection time 3 3 (UA / ml)

A before flying 42.7 5.46 1.09 <0.34 300

 After flight tt 7.85 1.28 0.34 460

B Before scattering 31.9 4.33 72.5 52.6 770

 After defeat 36.8 3.56 78.8 47.9 840

Before C defeat 15.1 ς 68.7 66.1 450

 After flying 20.3 1.32 64.3 49.7 330

Before D Fly 13.9 1.1 1 39.3 63.4 200

 After agility 1 B.4 0.81 31.9 54.7 120

E before flying 5.25 0.4 5. 0.34 <0.34 30

 8.33 0.46 <0.34 0.34 38

F Before scattering 6.64 0.39 0.34 0.34 26

 After flight tt 8.21 0.47 <0.34 0.34 27

G Before scattering 1.29 0.34 0.34 0.34 26

 After flying 4.02 0.34 <0.34 <0.34 30

H flying ΙΚ 1.99 0.41 26.5 36.1 220

 After flying tt 3.65 0.53 23.2 29.5 1 50

1 Scatter B 0.93 <0.34 54.6 51.7 1 30

 3.51 0.34 43.3 39.9 100

J hit tt before 3.55 0.68 0.55 0-34 96

 After flight tt 2.77 0.42 0.39 <0.34 78

K flying ttsij 1.2 <0.34 <0.34 <0.34 96

 2.72 0.34 0.34 0.34 110 after Tftlb 110 before scattering 0.95 0.39 1.3 1.8 13

 7t «¾t 2.5 0.51 1.45 2.38 18

M before K <0-34 0.34 <0.34 <0.34 36

 After flying κ 2.08 <0.34 0.34 <0.34 43

N flying Μ «ΰ 0.42 0.34 <0.34 0.34 22

 After flying 1.67 <0,34 0.45 <0.34 73

0 Before scattering 0.54 <0.34 28.1 27.2 180

 After flying 1.42 <0.34 27.2 26.3 160

P 0.38 <0.34 5.08 3.65 280

 After flying K 0.68 <0.34 4.49 3.02 240

0.34 0.34 0.34 0.34 5.0

 After scattering <0.34 0.34 <0.34 <0.34 <5.0

R flying MM 0.34 0.34 0.34 <0.34 53

 After scattering <0.34 0.34 0.34 <0,34 62

S flying Ms <0.34 <0.34 <0.34 0.34 420

 After flying tt 0.34 0-34 <0.34 <0.34 370

T fly 0.34 <0.34 <0.34 0.34 82

 <0.34 0.34 <0.34 <0.34 62 u Before flying <0.34 0.34 <0.34 0.34 18

 After scattering <0.34 0.34 <0.34 0.34 16

V flying Μ <0.34 0.34 0.79 0.81 180

After defeat 0.34 0.34 0.78 0.9 160 Based on the nucleotide sequence of the DD band determined in Example 7, primers 465-5 ′ (GAATCTCCCCGATCCCAAZ SEQ ID NO: 6), 465-3 ′ (ATAACCGTAAACACAAAGTAATACAGT ATTTZ SEQ ID NO: 7), and TaqMan probe 465-16SEQ (TGTTCCCAGTCACAGTAAA GATATTACCATCCTZ SEQ ID NO: 8) was designed, synthesized, and used for the quantitative reaction. TaqMan probe 465-16SEQ was used with its 5 'end labeled with FAM (6-carboxyfluorescein) and its 3' end with TAMRA (6-carboxy-tetramethy rhodamine). For type I, cDNA obtained by reverse transcription of 44 total RNAs using poly T (12-18 mers) as a primer was used. For the standard curve for calculating the copy number, the reaction was performed using a serial dilution of the plasmid p465-16 obtained in Example 7 as type III. The composition of the reaction mixture for monitoring PCR amplification is shown in Table 3. In addition, in order to correct the difference in cDNA concentration in the sample, the same quantitative analysis was performed for the / 3-actin (/ 3-actin) gene, and correction was performed based on the copy number of those genes to obtain the target gene (465 ) Was calculated.

 Table 3

 Reaction composition of ABI-PRISM 7700 (reaction volume per 1 liter) Sterile distilled water 25.66 (L)

 10x TaqMan Buffer A 5

25mM MgCl ₂ 7

 dATP (lOmM) 1.2

 dCTP (lOmM) 1.2

 dGTP (lOmM) 1.2

 dUTP (lOmM) 1.2

 Forward Primer (lOO ^ M) 0.15

 Reverse Primer (lOO ^ M) 0.15

 465 TaqMan probe (6.7 / M) 1.49

Am liTaq Gold (5U / D 0.25 AmpErase UNG (IL '/ L) 0.5

 Template solution 5

Table 4 shows the number (copy number) of “465” in each sample, corrected for the copy number of 50 i3-actin. For the correction, the average copy of 3-actin in all samples was determined, and the copy number of “465” in each sample was divided by the relative value of 3-actin in each sample when that was set to 1.

Table 4

Quantitative determination by ABI7700 (copy / ng RNA) beta actin correction data Recipient R JDS collection time Nozuno MU

 465

A before defeat 1 (mix? * O

B Fly WC Mae 09

 O

 C before IR 39

 ΙΙΚικ I Ο

D? ttlKIu ol

 3D Mr ι υ t

 7tf H 1 * ゥ .0 c c

 r * fO

 7KHX1! K yu

 Ό

 p u TKH H

 1

 1 TtffuH 3

 DO

 J 7 f 11

 71f nX x 1 (lx TTCnxB

 7ltHXl9E

 u 7nmH

 7ΠΡΙΧ1Κ

 M ffitfr front 4 * t1

 1

7TVHX19C 17 f

IN TtH B OD

 7TCHX19c * t 1 π TtHXH

 After the flight ??

P Before Defeat 13

 After defeat 42

Q Before scattering 64

 After scattering 55

R Before flying 37

 After defeat 5

S Before Defeat 39

 After scattering 28

T Before Defeat 48

 After defeat 33 u Before flying 75

 After flying 42

V Before scattering 47

After flying 32 A pairwise test was performed using this value. StatView software (Abacuus Concepts, Inc.) was used for the assay. The results showed that the expression of “465” was significantly higher in the pre-scatter group than in the post-scatter group (P value = 0.0151) when divided into groups before and after cedar pollen scatter (Fig. 2). The expression level of “465” in the group before pollen dispersal and the drop after dispersal was 57.9 ± 43.6 and 31.6 ± 19.2 copies / ng RNA (mean soil standard deviation), respectively.

 [Example 9] Chromosome mapping of "465"

 To determine the position of “465” on the human chromosome, chromosome mapping was performed using the radiation hybrid method (RH method). The RH method is based on the combination of hybrid cells (RH panel) obtained by irradiating human diploid cells with X-rays to physically cut chromosomes and fusing them with hamster-derived A23 cells. This is a method of determining the presence of the gene of interest by PCR and determining the position by linkage analysis with about 10,000 known markers (Hudson TJ et al., 1995, Science 270: 1945). -1954; Schuler GD et al., 1996, Science 274: 540-546; Stewart, EA and D. Cox, 1997, Radiation Hybrid Mapping. In Genome Mapping: A Practical Approach (ed. Paul Dear) .pp. 73-93. Oxford University Press, 0xford). The RH panel used Gene Bridge 4 (White Head Laboratory). 465-5 '(SEQ ID NO: 6) and 465-3' (SEQ ID NO: 7) were used as PCR primers. The PCR product was electrophoresed on a 4% agarose gel to check for the presence of a band. At this time, hamster chromosome-derived bands of different sizes were observed, but were treated as a background. As a result of mapping, “465” was found to be significantly linked to WI-3188 (GenBank Accession No. G04220), which is the best candidate on chromosome 3 ql3.

 [Example 10] Northern hybridization of "465"

Human Immune System MTN Blot II and Human Cancer Cell Line MTN Blot, which are membranes on which mRNAs prepared from various immune-related tissues and cancer cell lines are transcribed. Northern analysis was performed using (CLONTECH).

A DNA fragment of about 0.3 kb was excised from the plasmid into which the DD fragment of “465” was cloned using a restriction enzyme, and purified by agarose gel electrophoresis. The obtained fragment was labeled with ³² P using Random Primer Labeling Kit (TAKARA) and used as a probe. Using the Express Hybridization Solution (CL0NTECH), Northern Hybridization and membrane washing were performed as per the attached instructions. The washed membrane was exposed to an imaging plate, and an image was obtained using a Molecular Imager System (BIO-RAD). As a result, expression of about 9 kb mRNA was generally observed in the tissues and cell lines used. In immune-related tissues, relatively strong expression was observed, particularly in the spleen and lymph nodes, and in cancer cell lines, particularly strong expression was observed in chronic myeloid leukemia cells K-562 and lymphoblastic leukemia cells M0LT-4. Admitted. (Figure 3 and Figure 4)

 [Example 11] Cloning of "465" and analysis of nucleotide sequence

The sequence of the DD band obtained in Example 7 was 268 bp. Using the Marathon cDNA Amplification Kit (CLONTECH), create type II from human leukocyte mRNA, and use the kit-supplied API primer and 465-specific primer 465U (ATCTCCCCGATCCCAACTGTTCCZ sequence) based on the DD band sequence. No .: 9) PCR was performed. Further, the obtained amplified fragment was subjected to PCR by using AP2 hybridizing with the sequence in the adapter and primer 465GTRA (CTGTTCCCAGTCACAGTAMGATATZ SEQ ID NO: 10) with the obtained amplified fragment as type III. When the amplified fragment was subcloned and sequenced, a sequence of about 0.7 kb including the sequence of the DD band was found. Next, type III was prepared using the mRNA of chronic myeloid leukemia cell line K-562, which showed a relatively strong signal as a result of the above-mentioned Northern hybridization experiment. Was done. PCR was performed using the API primer and the “465” -specific primer 465t4 (ACTTCCCATTTTCCCTTTCCTTCAG / SEQ ID NO: 11). Further, the obtained amplified fragment was made into type III, and PCR was carried out using AP2 hybridizing with the sequence in the adapter and primer 465t5 (CCCTTTCCTTCAGC CCTTGG SEQ ID NO: 12). Subtract the amplified fragment When the sequence was determined by sequencing, a sequence of about 1.4 kb including the sequence of the DD band was found. Furthermore, using 465-specific primer 465t8 (AAAATCAACAGACAAAATCAGCTGCATTZ SEQ ID NO: 13) as a primer for cDNA production, type II was prepared using mRNA of chronic myeloid leukemia cell line K-562. Then, an extension experiment similar to the above was performed. PCR was performed using API primer 1 and “465” -specific primer 465t8. Furthermore, the obtained amplified fragment was converted into type III, and PCR was performed using AP2 and primer 465t9 (CATTCCCATCTMAATAGCACCAAGAAZ SEQ ID NO: 14) which hybridize with the sequence in the adapter. When the sequence was determined by subcloning the amplified fragment, a sequence of 1951 bp including the sequence of the DD band was found. This sequence is shown in SEQ ID NO: 1.

[Example 12] Analysis by 5 'RACE method 1

 Marathon-Ready cDNA (CL0NTECH) Bone Marrow was used as type II for 5 'RACE analysis. For the primary 5'RACE-PCR reaction, PCR was performed using the attached API Primer (CCATCCTAATACGACTC ACTATAGGGC SEQ ID NO: 15) and 465-yRl Primer (AGTCTCG TCMCAAATGGTTCTGGZ SEQ ID NO: 16) specific to “465”. The reaction was performed. For the second (nested) 5'RACE-PCR reaction, the attached AP2 Primer (ACTCACTATAGGGCTCGAGCGGC SEQ ID NO: 17) and 465-yR2 Primer specific to “465” (GACTATACATCTTGACTGCAC AGCZ SEQ ID NO: 18) Was used to perform a PCR reaction. The primary PCR reaction conditions were: 1 cycle at 94 ° C for 3 minutes, followed by 6 cycles at 94 ° C for 30 seconds, 60 and 30 seconds, and 68 minutes. For 1 cycle and then continuously at 4. The second is one cycle of `` 94 ° C for 3 minutes '' followed by one cycle of `` 72t: 5 minutes '' after 25 cycles of `` 94 ° C for 30 seconds, 60 ° C for 30 seconds, 72 minutes '', and then Continuously increased to 4. In both reactions, a reaction solution was prepared from the attached reaction reagent according to the attached manual using Advantage-GC cDNA Polymerase Mix (CLONTECH) as a Taq enzyme.

For gel electrophoresis, a 1.0% agarose gel was prepared, a sample was applied, and electrophoresed at a constant voltage of 100 V for 30 minutes. Then, electrophoresed by UV transillumination I got an image.

 [Example 13] Collection and sequencing of band excised by 5 'RACE analysis 1

 To determine the base sequence of the band obtained by 5 'RACE analysis, the amplified DNA fragment was excised from the gel, cloned with plasmid vector pT7Blue-T-Vector (Novagen), and the DNA fragment of about 800 bp Was obtained. Using a plasmid DNA, the nucleotide sequence of the DNA fragment was determined according to a conventional method. As a result, a further upstream sequence of about 600 bp was obtained. The sequence is shown in SEQ ID NO: 19.

 [Example 14] "Analysis by 5 'RACE method 2"

Marathon-Ready cDNA (CL0NTECH) Bone Marrow was used as type II for 5 'RACE analysis. For the primary 5'RACE-PCR reaction, PCR was performed using the attached API Primer (CCATCCTAATACGACTC ACTATAGGGC NO: 15) and the 465-yR4 Primer (CACTAAGG MTTTGCTMGGTCTGAZ SEQ ID NO: 20) specific to “465”. The reaction was performed. For the second (nested) 5'RACE-PCR reaction, the attached AP2 Primer (ACTCACTATAGGGCTCGAGCGGC / SEQ ID NO: 17) and 465-yR5 Primer specific to “465” (GAAGCAGGGCTTTTAGGAGGTG ATZ SEQ ID NO: 21) ) Was used to perform a PCR reaction. The 465-yR4 Primer and 465-yR5 Primer were designed based on the nucleotide sequences obtained from “Recovery of band excised by 5 ′ RACE analysis and sequencing 1”. The PCR reaction conditions were as follows: primary: one cycle of 943 minutes, followed by 35 cycles of 30 seconds at 94, 30 seconds at 60 ° C, and 68 minutes at 68 minutes, followed by 5 minutes at 68 minutes Was changed to 4 for 1 cycle and then continuously. The second is one cycle of "94 minutes at 3 minutes", followed by one cycle of "94: 30 minutes, 60 degrees Celsius, 30 seconds, 72 minutes" after 20 cycles, followed by one cycle of "721: 5 minutes", and then Continuously at 4. In both reactions, using the Advantage-GC cDNA Polymerase Mix (CLONTECH) as a Taq enzyme, reaction solutions were prepared from the attached reaction reagents according to the attached manual. For gel electrophoresis, a 1.0% agarose gel was prepared, 5 ^ 1 sample was applied, and electrophoresis was performed at 100 V constant voltage for 30 minutes. Thereafter, electrophoretic images were obtained by UV transillumination. [Example 15] Collection and sequencing of band excised by 5 'RACE analysis 2

To determine the base sequence of the band obtained by 5 'RACE analysis, the amplified DNA fragment was excised from the gel, and cloned with plasmid vector pT7Blue-T-Vector (Novagen) to obtain about 1 A plasmid carrying a kbp DNA fragment was obtained. Using a plasmid DNA, the nucleotide sequence of the DNA fragment was determined according to a conventional method. As a result, a further upstream sequence of about 900 bp was obtained. The sequence is shown in SEQ ID NO: 22. Summarizing these findings, the nucleotide sequence of “465” was finally 3442 bp. The nucleotide sequence of "465" is shown in SEQ ID NO: 23. Industrial applicability

 According to the present invention, a novel gene that can be used as an indicator of the response of T cells to antigen stimulation such as cedar pollen was provided. Using the expression of the gene of the present invention as an index, it has become possible to carry out a test for the presence or absence of a T cell response to pollen antigen, or a screening for a therapeutic drug candidate compound that suppresses the T cell response to antigen stimulation.

Claims

The scope of the claims 1. A nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO: 23.  2. A nucleic acid molecule comprising the protein coding region of the nucleotide sequence of SEQ ID NO: 23. 3. A DNA that specifically hybridizes to the nucleic acid molecule according to claim 1 or 2, and has a chain length of at least 15 nucleotides.  4. The method for detecting a nucleic acid molecule according to claim 1, wherein the DNA according to claim 3 is used.  5. a test method for an allergic disease,  (a) preparing T cells from a subject,  (b) preparing an RNA sample from the T cells,  (c) performing a hybridization on the RNA sample, using the labeled DNA of claim 3 as a probe,  (d) measuring the amount of RNA derived from a subject that hybridizes to the labeled DNA of claim 3, and comparing it with a control (in the case of a healthy subject).  6. A test method for an allergic disease,  (a) preparing T cells from a subject,  (b) preparing an RNA sample from the T cells,  (c) performing a reverse transcription reaction on the RNA sample to synthesize cDNA;  (d) a step of performing a polymerase chain reaction (PCR) using the cDNA as a type I and the DNA according to claim 3 as a primer,  (e) comparing the amount of DNA amplified by the polymerase chain reaction with a control (in the case of a healthy subject).  7. The method according to claim 6, wherein the polymerase chain reaction is performed by a PCR amplification monitor method. 8. The method according to claim 5, wherein the T cells are prepared from peripheral blood of the subject. The method described.  9. The method according to any one of claims 5 to 8, wherein the allergic disease is cedar pollinosis.  10. A method for screening a candidate therapeutic compound that suppresses the T cell antigen-stimulatory response,  (a) Step of administering a test compound to a model animal and stimulating it with a pollen antigen (b) preparing T cells from the model animal,  (c) preparing an RNA sample from the T cells,  (d) performing a hybridization on the RNA sample, using the labeled DNA of claim 3 as a probe.  (e) measuring the amount of the RNA derived from the T cells that hybridizes to the labeled DNA according to claim 3,  (f) selecting a compound that suppresses a decrease in the amount of RNA measured in step (e) as compared to a control (in the case where a test compound is not administered).  1 1. A method of screening for a candidate therapeutic compound that suppresses the T cell antigen-stimulatory response,  (a) administering a test compound to a model animal and stimulating with a pollen antigen, (b) preparing T cells from the model animal,  (c) preparing an RNA sample from the T cells,  (d) performing a reverse transcription reaction on the RNA sample to synthesize cDNA;  (e) performing a polymerase chain reaction (PCR) using the cDNA as a type I and the DNA according to claim 3 as a primer,  (f) selecting a compound that suppresses a decrease in the amount of DNA amplified in step (e) as compared to a control (in the case where no test compound is administered). 1 2. A method of screening for a candidate therapeutic compound that suppresses the T cell antigen-stimulatory response, (a) administering a test compound to a model animal,  (b) preparing lymphocytes from the model animal,  (c) stimulating the lymphocytes with a pollen antigen,  (d) separating T cells from the antigen-stimulated lymphocytes,  (e) preparing an RNA sample from the T cells,  (f) a step of performing hybridization on the RXA sample using the labeled DNA of claim 3 as a probe,  (g) a step of measuring the amount of RNA derived from the T cells that hybridizes to the labeled DNA of claim 3;  (h) selecting a compound that suppresses the decrease in the amount of RNA measured in step (g) as compared to a control (in the case where no test compound is administered).  13. A method of screening for a candidate therapeutic compound that suppresses the T cell antigen-stimulatory response,  (a) administering a test compound to a model animal,  (b) preparing lymphocytes from the model animal,  (c) stimulating the lymphocytes with a pollen antigen,  (d) separating T cells from the antigen-stimulated lymphocytes,  (e) preparing an RNA sample from the T cells,  (f) performing a reverse transcription reaction on the RNA sample to synthesize cDNA;  (g) performing a polymerase chain reaction (PCR) using the cDNA as a type I and the DNA according to claim 3 as a primer;  (h) selecting a compound that suppresses a decrease in the amount of DNA amplified in step (g) as compared to a control (in the case where no test compound is administered).  14. A method for screening a candidate therapeutic compound that suppresses the T cell antigen-stimulatory response, (a) preparing lymphocytes from a model animal or human, (b) stimulating the lymphocytes with a pollen antigen in the presence of a test compound,  (c) separating T cells from the antigen-stimulated lymphocytes,  (d) preparing an RNA sample from the T cells,  (e) a step of subjecting the labeled RNA sample to professional hybridization with the labeled DNA according to claim 3,  (f) measuring the amount of RNA derived from the T cells that hybridizes to the labeled DNA of claim 3,  (g) a step of selecting a compound that suppresses a decrease in the amount of RNA measured in step (II) compared to a control (in the case where no test compound is administered).  1 5. Τ A method for screening candidate therapeutic compounds that suppress the antigen-stimulated response of cells,  (a) preparing lymphocytes from a model animal or human,  (b) stimulating the lymphocytes with a pollen antigen in the presence of a test compound,  (c) separating T cells from the antigen-stimulated lymphocytes,  (d) preparing an RNA sample from the T cells,  (e) performing a reverse transcription reaction on the RNA sample to synthesize cDNA;  (f) performing a polymerase chain reaction (PCR) using the cDNA as a type I and the DNA according to claim 3 as a primer,  (g) selecting a compound that suppresses a decrease in the amount of DNA amplified in step (f) compared to a control (in the case where no test compound is administered).  16. A method for screening a candidate therapeutic compound that suppresses the T cell antigen-stimulatory response,  (a) stimulating the established T cells with a lymphocyte stimulating substance in the presence of the test compound, (b) preparing an RNA sample from the stimulated established T cells, (c) performing a hybridization on the RNA sample using the labeled DNA of claim 3 as a probe, (d) measuring the amount of RNA derived from the established T cell that hybridizes to the labeled DNA of claim 3,  (e) selecting a compound that suppresses the decrease in the amount of RNA measured in step (d) as compared to a control (in the case where no test compound is administered).  1 7. A method of screening for a candidate therapeutic compound that suppresses the T cell antigen-stimulatory response,  (a) stimulating the established T cells with a lymphocyte stimulating substance in the presence of the test compound, (b) preparing an RNA sample from the stimulated established T cells,  (c) performing a reverse transcription reaction on the RNA sample to synthesize cDNA;  (d) a step of performing a polymerase chain reaction (PCR) using the cDNA as a type I and the DNA according to claim 3 as a primer,  (e) selecting a compound that suppresses a decrease in the amount of DNA amplified in step (d) as compared to a control (in the case where no test compound is administered).  18. The method according to claim 10 or 11, wherein the T cells are prepared from peripheral blood of a model animal.  19. The method according to any of claims 12 to 15, wherein the lymphocytes are prepared from peripheral blood.  20. The method according to any one of claims 10 to 19, wherein the antigen is cedar pollen antigen.